Herbal compositions and methods for enhancing vital energy and athletic performance

ABSTRACT

Compositions, kits and methods are provided for enhancing vital energy and athletic performance, improving or restoring blood circulation, promoting mental acuity, reducing fatigue, and improving aerobic performance. In one embodiment, the composition comprises the herbal extracts of  Rhodiola crenulata  (root) and  Ginkgo biloba  (leaf). The composition can be used as a pharmaceutical or nutraceutical to promote mental concentration, and to promote aerobic and anaerobic performance by enhancing strength, endurance, muscle tissue oxygenation, and optimal oxygen consumption.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.61/045,717, filed Apr. 17, 2008, which application is incorporatedherein by reference.

BACKGROUND

In Chinese Medicine, physical exercise has a close relationship withvital energy (“qi”, also known as “chi”) health. In Chinese Medicinemovements of the muscles and the four limbs are believed to depend onthe power of the vital energy-qi. When the internal vital energy issufficient, the limbs and muscles are healthy and strong because theyare nourished by the blood and qi. If the internal vital energy isdeficient however, the muscles become weak, resulting in feelings oftiredness and general malaise.

Vital energy plays an important role in supporting physical activitiesof the body. During intense periods of physical exercise, especiallyendurance types, there are great demands on muscle energy and alsocardiovascular and pulmonary functions. In Chinese Medicine, these areall closely related to the vital energy-qi of the body. Lung qi isresponsible for pulmonary functions while heart qi is responsible forcardiovascular function. Physical activities, including the limbmovements, on the other hand, are mainly coordinated by spleen qi.Therefore, the body's physical performance is closely linked with havingan adequate amount of vital energy so that the organs can communicateand work efficiently with one another.

Intense physical exercise can easily exhaust the body's energy storesand consume vital energy. In order to maintain continuous performance inphysical exercise, it is important to pay attention to the recovery ofvital energy. Adequate rest is essential. If exhaustion is so severethat the body is unable to recover, an individual can develop a certaindegree of deficiency of qi and vital energy, which may manifest asfatigue, lack of energy and unsatisfactory performance of physicalactivities. If the deficiency is very severe or persists for a longtime, it can even affect a person's overall health because vital energyis also responsible for the majority of physiological activities in thebody.

Balance between vital energy health and physical exercise is veryimportant. If there is an optimal balance, physical exercise can benefitthe health of the whole body because it promotes blood and qicirculation, which in turn supports vital energy health. On the otherhand, if physical exercise is too intense, it can deplete the energyreserve of the body and exhaust the vital energy which over time maythen result in health problems. Therefore, to maintain health and reachoptimal physical performance, new methods of improving the body'srecovery after exercise are desirable, especially to improve therecovery of the vital energy.

SUMMARY

The present disclosure provides novel compositions, kits and methods forpharmaceutical or nutraceutical use in a mammal, preferably in a human.

In one aspect, compositions are provided, comprising an herbal extractof Rhodiola crenulata and an herbal extract of Ginkgo biloba. Thecomposition may further comprise one or more excipients. The compositionmay be a solid, liquid, or an aerosol.

In some embodiments, the herbal extract of Rhodiola crenulata comprisesat least about 0.40%, at least about 0.50%, at least about 0.60%, atleast about 0.70%, at least about 0.80%, or at least about 0.90% w/wsalidroside based on the total weight of the herbal extract.

In some embodiments, the herbal extract of Ginkgo biloba comprises atleast about 5.0%; at least about 6.0%, at least about 7.0%, at leastabout 8.0%, at least about 9.0%, at least about 10.0%, at least about11.0%, at least about 12.0%, at least about 13.0%; or at least about14.0% w/w flavonoids based on the total weight of the herbal extract.

In some embodiments, the herbal extract of Rhodiola crenulata comprisesabout 5-95% w/w of the total weight of the composition. In some of theembodiments, the herbal extract of Ginkgo biloba comprises about 5-50%w/w of the total weight of the composition. In some embodiments, theherbal extract of Rhodiola crenulata comprises about 50-95% w/w and theherbal extract of Ginkgo biloba comprises about 5-50% w/w of the totalweight of the composition.

In some embodiments, the herbal extract of Rhodiola crenulata is about90% w/w and the herbal extract of Ginkgo biloba is about 10% w/w of thetotal weight of the composition. In other embodiments, the herbalextract of Rhodiola crenulata is about 45-70% w/w and the herbal extractof Ginkgo biloba is about 5-10% w/w of the total weight of thecomposition.

In another aspect of the present disclosure, a method for improving orrestoring blood circulation in the body of a mammal is provided,comprising administering to a mammal in need thereof an effective amountof the composition described herein. The mammal is preferably a human,more preferably an athlete, and most preferably a professional athlete.

In yet another aspect, a method for promoting mental acuity in a mammalis provided, comprising administering to the mammal in need thereof aneffective amount of the composition described herein. The mammal may bea pet animal, but preferably a human.

In a further aspect of the disclosure, a method for reducing fatigue ina mammal is provided, comprising administering to the mammal in needthereof an effective amount of the composition of the presentdisclosure. The mammal is preferably a human.

In another aspect, a method for promoting aerobic and anaerobic sportsperformance in a mammal is provided, comprising administering to themammal in need thereof an effective amount of the composition disclosedherein. The mammal is preferably a human, more preferably an athlete,and most preferably a professional athlete.

In some embodiments, the administration of compositions disclosed hereinto a mammal is via an oral route. In some embodiments, the compositionis administered to a mammal at a dose of about 1-2600 mg/day, morepreferably about 400-2000 mg/day, even more preferably about 800-1600mg/day, even more preferably about 1200-1600 mg/day, and most preferablyabout 1000 mg/day. In some embodiments, multiple daily doses of 50, 100,200, 300, 400, 500, 600, 700, 800 or more mg per dose are provided.

In some embodiments, the composition is orally administered to a mammalat a daily dose of about 1-100 mg/kg, more preferably at about 5-50mg/kg, even more preferably about 10-30 mg/kg, and most preferably about20-25 mg/kg. Lower doses of about 1-6 mg/kg or about 1-5 mg/kg may beprovided. Multiple daily doses may include 2, 3, 4, 5, 6, 7, 8, 9, 10,or more doses. In various embodiments directed to oral administrationdisclosed herein, the mammal is a human.

In another aspect, a kit is provided, comprising: a container containingat least one dose of an effective amount of the composition disclosedherein. In some embodiments, the kit further comprises instructions ofhow to use the kit to improve or restore blood circulation, promotemental acuity, reduce fatigue, or promote aerobic performance. In someembodiments, the kit comprises more than one dose of an effective amountof the composition disclosed herein.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIG. 1 is a flow chart representing an extraction process for Rhodiolacreulata.

FIG. 2 is a flow chart representing an extraction process for Ginkgobiloba leaf.

FIG. 3 is a flow chart representing a manufacturing process disclosedherein.

FIG. 4 depicts graphs representing the effects of 7-week supplement withplacebo and RGC on changes from baseline in time to exhaustion (A) andVO_(2max) (B) in maximal incremental test. Data are expressed asmean±standard error mean (SEM) and analyzed using two-way repeatedmeasure ANOVA, followed by multiple group comparison. * P<0.05 vs.placebo values.

FIG. 5 depicts graphs representing the effects of 7-week supplement withplacebo (n=34) and RGC (n=33) on testosterone (A), cortisol (B), andratio of testosterone to cortisol (C) from baseline (stripped bars) toendpoint (filled bars). Data are expressed as mean±standard deviation(SD) and analyzed using two-way repeated measure ANOVA, followed bymultiple group comparison. # P<0.05: vs. baseline values.

FIG. 6 depicts a graph of a MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Assayrepresenting the effects of RG, Rhodiola crenulata, and Ginkgo on theviability of L6 cells under hypoxic conditions. Cell viability wasmeasured by the MTT assay. Each histogram represents the mean with S.D.of 6 determinations. *, significantly different from vehicle at P<0.05;**, significantly different from vehicle at P<0.01.

FIG. 7 depicts a graph of a LDH (lactate dehydrogenase) Assayrepresenting the effects of RG, Rhodiola crenulata, and Ginkgo biloba onthe LDH leakage of L6 cells under hypoxic conditions. LDH leakage wasmeasured by the LDH assay. Each histogram represents the mean with S.D.of 5-6 determinations. *, significantly different from vehicle atP<0.05; **, significantly different from vehicle at P<0.01.

FIG. 8 depicts a graph of a SOD (superoxide dismutase) Antioxidant Assayrepresenting the effects of RG, Rhodiola crenulata, and Ginkgo biloba onthe SOD activity of L6 cells under hypoxic conditions. SOD activity wasmeasured by the SOD assay. Each histogram represents the mean with S.D.of 5-6 determinations. *, significantly different from vehicle atP<0.05; **, significantly different from vehicle at P<0.01.

FIG. 9 depicts a graph representing the effects of RG, Rhodiolacrenulata, and Ginkgo biloba on the SOD activity of L6 cells underhypoxic conditions. Group 1—80% Rhodiola crenulata+20% Ginkgo biloba(e.g. total 80 ug/ml), Group 2—80% Rhodiola crenulata (e.g. total 64ug/ml), Group 3—100% Rhodiola crenulata (e.g. total 80 ug/ml), Group 4(“RG” group)—90% Rhodiola crenulata+10% Ginkgo biloba (e.g. total 80ug/ml); Group 5—90% Rhodiola crenulata (e.g. total 72 ug/ml), Group6—Vit. C as control group, Group 7—Vehicle, Group 8—Control. LDH leakagefor each group was measured by the LDH assay. Each histogram representsthe mean with S.D. of 5-6 determinations. *, significantly differentfrom vehicle at P<0.05; # significantly different from 4 at P<0.05.

FIG. 10 depicts a graph representing the effects of Group 1 to Group 7on the SOD activity of L6 cells under hypoxic conditions. Group 1—80%Rhodiola crenulata+20% Ginkgo biloba (e.g. total 80 ug/ml), Group 2—80%Rhodiola crenulata (e.g. total 64 ug/ml), Group 3—100% Rhodiolacrenulata (e.g. total 80 ug/ml), Group 4 (“RG” group)—90% Rhodiolacrenulata+10% Ginkgo biloba (e.g. total 80 ug/ml); Group 5—90% Rhodiolacrenulata (e.g. total 72 ug/ml), Group 6—Vit. C as control group, Group7—Vehicle, Group 8—Control. SOD activity was measured by the SOD assay.Each histogram represents the mean with S.D. of 5-6 determinations. *,significantly different from vehicle at P<0.05. # significantlydifferent from 4 at P<0.05.

DETAILED DESCRIPTION

The present disclosure provides novel compositions for nutraceutical orpharmaceutical use in a mammal, preferably in a human. In someembodiments, the composition comprises the herbal extract of Rhodiolacrenulata at about 50-90% w/w and the herbal extract of Ginkgo biloba atabout 5-30% w/w based on the total weight of the extract combination.Any combination of proportions of the herbal extracts of Rhodiolacrenulata and Gingko biloba are envisioned to be encompassed by thecompositions disclosed herein. In one embodiment, the composition is acombination of Rhodiola crenulata (root) at about 90% w/w and Ginkgobiloba (leaves) at about 10% w/w based on the total weight of the herbalextract components of the composition. The percentages provided hereinrefer to the w/w ratio based on the total weight of the herbal extractportion of the composition and do not include any excipients of extraingredients added to a formulation.

In one aspect, compositions are provided which comprise an herbalextract of Rhodiola crenulata and an herbal extract of Ginkgo biloba.The combination of these herbal extracts may enhance their functionscompared to that when administered alone. Therefore, the combinationsynergizes the activity of the herbal extracts, as well as decreases oneor more toxic effects of the constituent herbs. Compositions of thepresent disclosure may be, for example, solid, liquid, or aerosolformulations comprising one or more of the extracts as disclosed herein.Compositions of the disclosure may also comprise other components, forexample, vitamins, pharmaceuticals or excipients.

Folium Ginkgo consists of the dried whole leaf of Ginkgo biloba. Ginkgobiloba is thought to have neuroprotective properties; however, the exactneuroprotective mechanism of Ginkgo biloba is not known, but Ginkgobiloba herbal extract components include flavone glycosides (which ismade up of quercetin, kaempferol, rutin and myricetin) as well asterpene lactones (ginkgolides A and B), all of which decrease freeradical release. In addition, terpene lactones have been shown toimprove blood flow and reduce thrombus formation by inhibitingplatelet-activating factor. Without being bound by any one theory, onemechanism of action by which compounds of the disclosure functions is byremoving free radicals which cause oxidative damage to tissue. Forexample, Ginkgo biloba may reduce or prevent cell membrane lipidperoxidation, decrease oxidative damage to red blood cells and protectnerve cells. Thus, Ginkgo biloba herbal extract may benefit any cell ortissue that can be damaged by free radicals. In one embodiment, theGinkgo biloba herbal extract portion reduces or prevents oxidativedamage in the central nervous system and vasculature by improving bloodflow and circulation.

Further, Ginkgo biloba is a well-known memory-improving agent that hasbeen widely used in aged people and patients with memory problems (Birkset al., Cochrane Database Syst. Rev. 18:CD003120, (2007); Schulz,Phytomedicine 10(S): 74-79, (2003)). Moreover, Ginkgo biloba extractsare also used for preventing acute mountain sickness (Gertsch et al.,High Alt. Med. Biol. 3:29-37, (2002); Gertsch et al., BMJ. 328:797,(2004)) and relieving the symptoms of intermittent claudication (Pittler& Ernst, Am. J. Med. 108:276-281, (2000)), suggesting that Ginkgo bilobamay have a capacity to improve mental conditions and physical workperformance.

The Rhodiola used in the compositions of the present disclosure can beisolated from any species of Rhodiola. Preferably, the Rhodiola used isan extract isolated from the dried root and rhizome of Rhodiolacrenulata. Rhodiola is a high altitude growing plant and there are about200 species, including R. rosea and R. crenulata (Kelly, Altern. Med.Rev. 6:293-302, (2001); Ming et al., Phytother. Res. 19:740-743,(2005)). Rhodiola crenulata is a Tibetan plant which grows at altitudesgreater than 3500 meters. Rhodiola crenulata is an adaptogen which helpsthe body adapt to and resist a variety of physical, chemical, andenvironmental stresses. Therefore, in various embodiments, compositionsof the disclosure may be used to increase energy, stamina, strength, andmental capacity. In further embodiments, compositions of the disclosuremay help to reduce fatigue and prevent altitude sickness. In oneembodiment, compositions of the disclosure administered to a mammalcomprise Rhodiola crenulata extract. One bioactive ingredient ofinterest in Rhodiola crenulata is salidroside. Rosavins is anotherbioactive constituent identified from the plant. Salidroside and/orrosavins can be used as references for determination of the quality ofthe preparations.

Furthermore, one aspect of the disclosure comprises a compositioncomprising a combined preparation from Rhodiola crenulata and Ginkgobiloba leaf. Such compositions may possess robust adaptogenic andergogenic effects. Such compositions can be manufactured in variousformulations, which are administered to a mammal to promote resilienceto environmental stressors such as increased oxygen demand or reducedoxygen availability, pollutants, time zone changes, lack of sleep, orenhance energy production.

Rhodiola crenulata and Ginkgo biloba may be extracted with alcohol,water, or alcohol/water and the extracts can be concentrated, and driedto solid form, such as in the form of a powder. Each may undergo asingle, or alternatively, double extraction process. In a preferredembodiment, Rhodiola crenulata (root) is extracted with alcohol(ethanol), concentrated, and dried to yield yellowish brown powder withthin odor and bitter taste. In a preferred embodiment, Rhodiolacrenulata (root) may go through an extraction process twice, each timewith alcohol and water. Ginkgo biloba (leaf) may be extracted withwater/alcohol, concentrated, and dried to yield light brownish yellowpowder with thin odor and bitter taste. One of skill in the art willrecognize that multiple processes of preparing plant extracts and can beused for the present disclosure, in addition to the particular processesdisclosed herein. See, e.g., U.S. Pat. No. 6,996,919.

In some embodiments, a composition of the disclosure comprises an herbalextract of Rhodiola crenulata containing a desired amount of about0.01%, about 0.05%, about 0.10%, about 0.15%, about 0.20%, about 0.25%,about 0.30%, about 0.35%, about 0.40%, about 0.45%, about 0.50%, about0.55%, about 0.60%, about 0.65%, about 0.70%, about 0.75%, about 0.80%,about 0.85%, about 0.90%, about 0.95%, about 1.00%, about 1.05%, about1.10%, about 1.15%, about 1.20%, about 1.25%, about 1.30%, about 1.35%,about 1.40%, about 1.45%, about 1.50%, about 1.55%, about 1.60%, about1.65%, about 1.70%, about 1.75%, about 1.80%, about 1.85%, about 1.90%,about 1.95%, or about 2.00% salidroside w/w of the total weight of theextract. In some embodiments, compositions of the present disclosurecomprise an extract of Rhodiola crenulata comprising 1-5% salidrosidew/w of the total weight of the extract. More preferably, the extractscomprise 1-2% salidroside, and most preferably, the extracts comprise1.2-1.7% salidroside w/w of the total weight of the extract.

In some embodiments, a composition of the disclosure comprises an herbalextract of Ginkgo biloba comprising not less than about 0.5%, about1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about4.0%, about 4.5%, about 5.0%, about 6.0%, about 6.5%, about 7.0%, about7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%, about10.5%, about 11.0%, about 11.5%, about 12.0%, about 12.5%, about 13.0%,about 13.5%, about 14.0%, about 14.5%, about 15.0%, about 15.5%, about16.0%, about 16.5%, about 17.0%, about 17.5%, about 18.0%, about 18.5%,about 19.0%, about 19.5%, about 20.0%, about 20.5%, about 21.0%, about21.5%, about 22.0%, about 22.5%, about 23.0%, about 23.5%, about 24.0%,about 24.5%, about 25.0%, about 25.5%, about 26.0%, about 26.5%, about270.0%, about 27.50%, about 28.0%, about 28.50%, about 29.0%, about29.5%, about 30.0%, about 30.5%, about 31.0%, about 31.5%, about 32.0%,about 32.5%, about 33.0%, about 33.5%, about 34.0%, about 34.5%, about35.0%, about 35.5%, about 36.0%, about 36.5%, or about 37.0% flavonoidsw/w of the total weight of the extract. Preferably, a composition of thedisclosure comprises an herbal extract of Ginkgo biloba comprising20-30% flavonoids w/w of the total weight of the extract. Even morepreferably, a composition of the disclosure comprises an herbal extractof Ginkgo biloba comprising 23-25% flavonoids w/w of the total weight ofthe extract. Most preferably, a composition of the present disclosurecomprises an herbal extract of Ginkgo biloba comprising not less than24% flavonoids w/w of the total weight of the extract.

In some embodiments, a composition of the present disclosure comprisesan herbal extract of Ginkgo biloba comprising no less than about 0.5%,about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%,about 4.0%, about 4.5%, about 5.0%, about 6.0%, about 6.5%, about 7.0%,about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%,about 10.5%, about 11.0%, about 11.5%, about 12.0%, about 12.5%, about13.0%, about 13.5%, about 14.0%, about 14.5%, or about 15.0% ofbilobalide (C₁₅H₁₈O₈), ginkgolide A (C₂₀H₂₄O₉), ginkgolide B(C₂₀H₂₄O₁₀), ginkgolide C (C₂₀H₂₄O₁₁), referred to as total terpenelactones, of the total weight of the Ginkgo biloba extract. Preferably,the compositions of the present disclosure comprise Ginkgo bilobaextracts comprising no less than 2-10% lactones w/w of the total weightof the extract. More preferably, the compositions of the presentdisclosure comprise Ginkgo biloba extracts comprising no less than 4-9%lactones w/w of the total weight of the extract. Even more preferably,the compositions of the present disclosure comprise Ginkgo bilobaextracts comprising no less than 5-8% lactones w/w of the total weightof the extract. In yet other preferred embodiments, the compositionscomprise Ginkgo biloba extracts no less than 6% lactones w/w of thetotal weight of the extracts. Most preferably, the compositions of thepresent disclosure comprise Ginkgo biloba extracts comprising no lessthan 5% lactones w/w of the total weight of the extract.

In some embodiments, the compositions of the present disclosure comprisean herbal extract of Rhodiola crenulata comprising about 5-95%, about30-95%, about 40-95%, about 50-95%, about 60-95%, or about 60-90% w/wbased on the total weight of the composition. In some embodiments, thecompositions of the present disclosure comprise an herbal extract ofRhodiola crenulata comprising about 5%, about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, or about 95% w/w of the total weight of the composition.

In some embodiments, the compositions of the present disclosure comprisean herbal extract of Ginkgo biloba comprising about 5-50%, about 5-40%,about 5-30%, about 5-20% w/w of the total weight of the composition. Insome embodiments, the compositions of the present disclosure comprise anherbal extract of Ginkgo biloba comprising about 1.0%, about 1.5%, about2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%, about 10.5%,about 11.0%, about 11.5%, about 12.0%, about 12.5%, about 13.0%, about13.5%, about 14.0%, about 14.5%, about 15.0%, about 15.5%, about 16.0%,about 16.5%, about 17.0%, about 17.5%, about 18.0%, about 18.5%, about19.0%, or about 20.0% w/w of the total weight of the composition.

After extraction, individual components of the herbal extracts can bemixed into a formulation which can be provided to humans and otheranimals. In some embodiments, this formulation may be an oral dosageform, including, but not limited to, a pill or tablet. In still furtherembodiments, these formulations contain 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 or more mg ofsalidroside derived from Rhodiola crenulata. In still furtherembodiments, these formulations contain 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5%salidroside. In some embodiments, these formulations also contain 1.5,1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 or more mg of flavonoid derived fromGinkgo biloba. In still further embodiments, these formulations contain0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%,1.3%, 1.4% or 1.5% flavonoid.

In some embodiments, the herbal components of the composition containminimal amounts of water. In some embodiments the herbal componentscontain less than 0.5% of water by weight. In other embodiments, theherbal components of the composition contain less than 0.1% water byweight.

The compositions of the disclosure may further comprise physiologicallyor pharmaceutically acceptable excipients. Excipients are selected toprovide formulations for specific routes of administration and/orpreferred types of administration, e.g., administration by tablet,capsule, or liquid dose.

For oral administration, the inventive compositions may optionally beformulated by mixing the herbal ingredients in combination withphysiologically or pharmaceutically acceptable carriers that are wellknown in the art. Such carriers enable the herbal ingredients to beformulated as tablets, pills, dragees, capsules, emulsions, lipophilicand hydrophilic suspensions, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by an individual or apatient to be treated.

In one embodiment, the inventive composition is contained in capsules.Capsules suitable for oral administration include push-fit capsules madeof gelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredients in admixture with filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers.

In some embodiments, a capsule contains about 50-1000 mg, about 100-800mg, about 150-600 mg, or about 200-400 mg of a mixture of herbalextracts of Rhodiola crenulata and Ginkgo biloba. In other embodiments,each capsule contains about 50 mg, about 100 mg, about 150 mg, about 200mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950mg, or about 1000 mg of a mixture of the herbal extracts of Rhodiolacrenulata and Ginkgo biloba. In other embodiments, each capsule containsabout 50 mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg,about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg,about 300 mg, about 325 mg, about 350 mg, or about 400 mg of a mixtureof the herbal extracts of Rhodiola crenulata and Ginkgo biloba. In yetother embodiments, each capsule contains about 100 mg, about 110 mg,about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg,about 170 mg, about 180 mg, about 200 mg, about 210 mg, about 220 mg,about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg,about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg,about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg,about 380 mg, about 390 mg, or about 400 mg of a mixture of the herbalextracts of Rhodiola crenulata and Ginkgo biloba. In yet otherembodiments, each capsule contains about 1000 mg, 100-800 mg, 150-600mg, or 200-400 mg of a mixture of herbal extracts of Rhodiola crenulataand Ginkgo biloba.

It is understood that these and other alternate embodiments of thedisclosure may include capsules formed of materials besides gelatin suchas vegetarian based capsules made from hydroxypropylmethylcellulose.

Optionally, the inventive composition for oral use can be obtained bymixing the inventive composition with a solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are, in particular, fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

For buccal administration, the inventive compositions may take the formof tablets or lozenges formulated in conventional manner. Each tablet orlozenge may contain about 50-1000 mg, about 100-800 mg, about 150-600mg, or about 200-400 mg of a mixture of herbal extracts of Rhodiolacrenulata and Ginkgo biloba. In some embodiments, each tablet or lozengecontains about 50 mg, about 100 mg, about 150 mg, about 200 mg, about250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about500 mg, about 550 mg, or about 600 mg of a mixture of the herbalextracts of Rhodiola crenulata and Ginkgo biloba. In other embodiments,each tablet or lozenge contains about 50 mg, about 75 mg, about 100 mg,about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg,about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, orabout 400 mg of a mixture of the herbal extracts of Rhodiola crenulataand Ginkgo biloba. In yet other embodiments, each tablet or lozengecontains about 100 mg, about 110 mg, about 120 mg, about 130 mg, about140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about250 mg, about 260 mg, about 270 mg, about t 280 mg, about 290 mg, about300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, or about400 mg of a mixture of the herbal extracts of Rhodiola crenulata andGinkgo biloba. In yet other embodiments, each tablet or lozenge containsabout 1000 mg, 100-800 mg, 150-600 mg, or 200-400 mg of a mixture ofherbal extracts of Rhodiola crenulata and Ginkgo biloba.

For administration by inhalation, the inventive composition for useaccording to the present disclosure may be delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas, or from propellant-free, dry-powder inhalers. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

Each dosage unit contains about 50-1000 mg, about 100-800 mg, about150-600 mg, or about 200-400 mg of a mixture of herbal extracts ofRhodiola crenulata and Ginkgo biloba. In some embodiments, each dosageunit contains about 50 mg, about 100 mg, about 150 mg, about 200 mg,about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg,about 500 mg, about 550 mg, or about 600 mg of a mixture of the herbalextracts of Rhodiola crenulata and Ginkgo biloba. In other embodiments,each dosage unit contains about 50 mg, about 75 mg, about 100 mg, about125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, or about400 mg of a mixture of the herbal extracts of Rhodiola crenulata andGinkgo biloba. In yet other embodiments, each dosage unit contains about100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about150 mg, about 160 mg, about 170 mg, about 180 mg, about 200 mg, about210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about260 mg, about 270 mg, about t 280 mg, about 290 mg, about 300 mg, about310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about360 mg, about 370 mg, about 380 mg, about 390 mg, or about 400 mg of amixture of the herbal extracts of Rhodiola crenulata and Ginkgo biloba.In yet other embodiments, each dosage unit contains about 1000 mg, about100-800 mg, about 150-600 mg, or about 200-400 mg of a mixture of herbalextracts of Rhodiola crenulata and Ginkgo biloba.

Methods of Use

In another aspect of the disclosure, a method of improving or restoringblood circulation is provided. The method of improving or restoringblood circulation comprises administering to a mammal in need thereof,an effective amount of any of the inventive compositions describedabove. The mammal is preferably a human.

In yet another aspect of the disclosure, a method of promoting mentalacuity in a mammal is provided. The method comprises administering to amammal in need thereof an effective amount of any of the inventivecompositions described above. The mammal is preferably a human. Byadministering a composition of the disclosure, mental acuity andconcentration are increased.

In another aspect of the disclosure, a method of reducing fatigue in amammal is provided. One method of reducing fatigue comprisesadministering to a mammal in need thereof, an effective amount of any ofthe inventive compositions described above. The mammal is preferably ahuman. Fatigue may be the result of exertion in a stressful environment,such as, for example, physical exertion at high altitudes, or may beresult of exercise regimes, or extraordinary physical performance.Fatigue may also be the result of performance in a stressful environmentsuch as a fast paced workplace, or travel across time zones, which canbe experienced in a non-athletic work situation.

In another aspect of the disclosure, a method of promoting aerobic andanaerobic performance in a mammal is provided. One such method ofpromoting aerobic and/or anaerobic performance comprises administeringto a mammal in need thereof, an effective amount of any of the inventivecompositions described above. The mammal is preferably a human. Aerobicperformance may be related to exercise, i.e. aerobic exercise or sportsperformance. Anaerobic performance may be related to high intensity, lowduration exertions (e.g., power lifting). Promotion of aerobic and/oranaerobic performance may also be directed towards enhancing muscletissue and optimizing performance from toned muscle mass. Promotion ofaerobic and/or anaerobic performance may also be directed towardsimproving the strength of muscle mass, or endurance of muscles oroverall aerobic endurance, i.e. distance running, skiing, or swimming.

In some of the methods of the present disclosure, a composition isadministered orally. In some of the embodiments of the methods of thedisclosure, the composition is administered via inhalation or aerosol.The amount of the inventive composition administered will be dependenton the subject being treated, the subject's weight, the manner ofadministration, and/or the judgment of the prescribing physician.Generally, however, the dosage of the inventive composition will beabout 0.01 mg/kg/day to about 1000 mg/kg/day, about 0.01 mg/kg/day toabout 500 mg/kg/day, about 1 mg/kg/day to about 600 mg/kg/day, about 5mg/kg/day to about 500 mg/kg/day, about 7 mg/kg/day to about 300mg/kg/day, about 10 mg/kg/day to about 150 mg/kg/day, or about 10mg/kg/day to about 100 mg/kg/day. In some embodiments, the dosage of theinventive composition is about 2-100 mg/kg/day, 5-50 mg/kg/day, 7-40mg/kg/day, or 8-30 mg/kg/day.

Further, the amount of the herbal extracts administered in the inventivecompositions can be about 0.01 mg/kg/day to about 200 mg/kg/day, about0.01 mg/kg/day to about 100 mg/kg/day, about 1 mg/kg/day to about 50mg/kg/day, about 5 mg/kg/day to about 30 mg/kg/day, about 7 mg/kg/day toabout 20 mg/kg/day, or about 10 mg/kg/day to about 15 mg/kg/day. In someembodiments the daily amount of the herbal extracts administered in theinventive compositions are about 0.1 mg/kg/day, about 0.5 mg/kg/day,about 1.0 mg/kg/day, about 1.5 mg/kg/day, about 2.0 mg/kg/day, about 2.5mg/kg/day, about 3.0 mg/kg/day, about 4.0 mg/kg/day, about 5.0mg/kg/day, about 6.0 mg/kg/day, about 7.0 mg/kg/day, about 8.0mg/kg/day, about 9.0 mg/kg/day, about 10.0 mg/kg/day, about 11.0mg/kg/day, about 12.0 mg/kg/day, about 13.0 mg/kg/day, about 14.0mg/kg/day, about 15.0 mg/kg/day, about 16.0 mg/kg/day, about 17.0mg/kg/day, about 18.0 mg/kg/day, about 19.0 mg/kg/day, about 20.0mg/kg/day, about 21.0 mg/kg/day, about 22.0 mg/kg/day, about 23.0mg/kg/day, about 24.0 mg/kg/day, about 25.0 mg/kg/day, about 26.0mg/kg/day, about 27.0 mg/kg/day, about 28.0 mg/kg/day, about 29.0mg/kg/day, about 30.0 mg/kg/day, about 31.0 mg/kg/day, about 32.0mg/kg/day, about 33.0 mg/kg/day, about 34.0 mg/kg/day, about 35.0mg/kg/day, about 36.0 mg/kg/day, about 37.0 mg/kg/day, about 38.0mg/kg/day, about 39.0 mg/kg/day, or about 40.0 mg/kg/day.

In some embodiments, the compositions are administered as tablets onceor twice a day. In some embodiments, more than one tablet isadministered at the same time. In some embodiments, the compositions areadministered as lozenges once or twice a day. In some embodiments, morethan one lozenge is administered at the same time. In some embodiments,the compositions are administered as capsules once or twice a day. Insome embodiments, more than one capsule is administered at the sametime. In some embodiments, the compositions are administered as liquiddoses once or twice a day. In some embodiments, more than one liquiddose is administered at the same time. In some embodiments, thecompositions are administered by inhalation once or twice a day.

In some embodiments of the disclosure, the composition is administeredto a human at a dose of about 1-2600 mg/day, more preferably about400-2000 mg/day, even more preferably about 800-1600 mg/day, even morepreferably about 1200-1600 mg/day, and most preferably about 1000mg/day. In some embodiments, multiple daily doses of 50, 100, 200, 300,400, 500, 600, 700 or more mg per dose are provided.

In some embodiments of the disclosure, the composition is orallyadministered to a human at a daily dose of about 1-100 mg/kg, morepreferably at about 5-50 mg/kg, even more preferably about 10-30 mg/kg,and most preferably about 20-25 mg/kg. Lower doses of about 1-6 mg/kg orabout 1-5 mg/kg may be provided. Multiple daily doses may include 2, 3,4, 5, 6, 7, 8, 9, 10, or more doses.

For example, to enhance aerobic performance, the dosage of the inventivecomposition is about 10-100 mg/kg, 20-60 mg/kg, or 30-50 mg/kg once ortwice a day. In some embodiments of the methods of the disclosure, thecomposition is administered to a mammal at a dose of about 1-100 mg/kg,about 1-60 mg/kg, about 1-30 mg/kg, about 1-15 mg/kg, about 1-6 mg/kg orabout 1-5 mg/kg once or twice a day. In other embodiments of the methodsof the disclosure, the composition is administered to a mammal at a doseof about 10-200 mg/kg, 20-100 mg/kg, or 40-80 mg/kg once or twice a day.

The inventive composition may also be administered in combination withanother therapeutic agent (e.g., Losartan, Simvastin, Ramipril, Aspirin,TPA and the like) or nutritional supplement (e.g., Lingzhi, green tea,and/or vitamins) to prevent or treat conditions described aboveadditively or synergistically. The inventive composition may beadministered prior to, concomitantly with, or subsequently to theadditional therapeutic agent or nutritional supplement.

Kits

The present disclosure also provides a kit or assembly of kitscontaining the inventive composition. A kit may contain the compositionwhich comprises Rhodiola crenulata and Ginkgo biloba in a uniform dosageform in a vessel. A kit may also contain a single dosage form ormultiple dosage forms. A kit may further comprise a dispensing tool,e.g. a pump, a cup, a spoon, a pipette or eyedropper, and the like. Akit may further comprise instruction as to how to use the kit to promotemental acuity, promote aerobic performance by enhancing strength,endurance, muscle tissue oxygenation, and optimal oxygen consumption,decrease fatigue, and/or promote or restore blood circulation.Instructions may be in a printed form.

Methods of Preparation

Rhodiola crenulata Extraction Process.

A schematic version of one process of this extraction process is shownin FIG. 1. The Rhodiola crenulata raw herb is weighed and ground intocoarse powder. The powder is put into an extraction chamber. Eight-timesthe total weight of the coarse powder of 50% ethanol is added and themixture is heated under reflux twice (the first time is for 2 hours andthe second time is for 1.5 hours) at temperature 80-85° C. and steampressure 0.1-0.2 MPa. The condensate is then filtered by the 120-meshfilter to discard the residue. The ethanol is then recollected from thefiltrate by evaporation at 70° C. and steam pressure 0.15 MPa. Theconcentration process is ended when the relative density is 1.2-1.3 at60° C. The concentrated extraction paste is transferred to the 300,000grade clean area and then dried in vacuum of −0.07 MPa at 80° C. andsteam pressure 0.2 MPa. The dry paste is finally ground into powder withthe extraction yield of 10% and stored in the 300,000 grade clean area.The ethanol used in the extraction process may be collected.

Ginkgo biloba Leaf Extraction Process.

A schematic version of one process of this extraction process is shownin FIG. 2. The Ginkgo biloba leaf raw herb is weighed and ground intocoarse powder. The powder is then put into an extraction chamber. Theherbal powder is heated under reflux twice with 70% ethanol. The firsttime: add 8:1 (w/w) 70% ethanol to the herb and heat under reflux for 2hours. The second time:add 6:1 (w/w) 70% ethanol to the herb and heatunder reflux for 1.5 hours. The temperature is 80-85° C. and steampressure 0.1-0.2 MPa. After the reflux, the condensate is filtered undernormal pressure and 120-mesh, and the residue discarded. Ethanol isrecollected from the filtrate and concentrated again, at temperature 70°C., steam pressure 0.15 MPa. The concentration process is stopped whenthe relative density reaches 1.15-1.2 (at 60° C.). Then 5:1 (w/w)purified water is added to the filtrate and left ot stand still for 24hours. The filtrate is run on the polyamide column (filtrate:Polyamide=1:4). Purified water is added to wash the column until theeluate becomes clear. After that, 25% ethanol is added and then 80%ethanol is added to wash the column. The eluate is collected andconcentrated at 70° C. and steam pressure 0.15 MPa. The concentratedextraction paste is transferred to the 300,000 grade clean area and thendried in vacuum of −0.07 MPa at 80° C. and steam pressure 0.2 MPa. Thedry paste is finally ground into powder with the extraction yield 10%and stored in the 300,000 grade clean area.

EXAMPLES Example 1 Analysis of salidroside by High Performance LiquidChromatography

Sample Preparation:

In liquid form: 20 ml of the sample solution is added to 25 ml methanolin a volumetric flask and is sonicated for 10 minutes. Afterwards,methanol is slowly added until the lowest part of the meniscus reachesthe graduation mark of the flask and is then mixed thoroughly in theflask. Lastly, the solution is filtered with a 0.45 um filter.

In solid form: More than 20 tablets or capsules are ground into apowder. A given mass of the powder (accurate to 0.001 g) is put into a50 ml volumetric flask. Methanol is added to the powder and the mixtureis sonicated for 10 minutes. Next, methanol is added to the solutionuntil its lowest meniscus reaches the graduation mark of the flask. Thesolution is then centrifuged at 300 rpm for 3 minutes. The supernatentis collected and filtered with a 0.45 um filter.

HPLC Analysis:

A C18 column of 4.6 mm×250 (length) mm, and 5 um beads is used. Theanalysis is performed at room temperature. The mobile phase is 0.02mol/L methanol in sodium acetate. Sample volume is 10 ul and flow rateis 1.0 ml/min. Detection is performed using a UV spectrophotometer at awavelength of 215 nm. The Standard curve is prepared by analyzingdifferent concentrations of a salidroside standard solution (0 ug/ml,0.01 ug/ml, 0.02 ug/ml. 0.05 ug/ml, 0.2 ug/ml, and 0.5 ug/ml) by HPLC.The peak height/peak area comparison is used to set up the standardcurve.

The collected data is analyzed using the following equation:

${X = \frac{h_{1} \times C \times V}{h_{2} \times m \times 1000}},$

where X=amount of Salidroside in the sample (mg/g), h₁=Peak height/areaof the sample solution; C=concentration of standard solution u/ml;V=volume of the sample solution; h₂=peak height/area of the standardsolution, and; m=mass of the sample. X is corrected to 3 significantfigures. The expected recovery rate is between 91.7% and 98.6%. For someherbal extractions, the absolute deviation of two individual testresults (repeated under identical conditions) should not exceed 10% oftheir average values.

Example 2 Analysis of Flavonoids by HPLC

Sample Preparation:

A 0.5 g sample is weighed and 25 ml ethanol is added and then mixed byshaking. The solution is then sonicated for 20 minutes and then standswithout agitation for a short period. One ml of the supernatant (clearfluid) is removed and added to an evaporation dish. One gram ofpolyamide powder is added. The ethanol from the solution is evaporatedby placing the evaporation dish over a water bath set above roomtemperature. After evaporation, the residue is added to a chromatographycolumn. 20 ml of benzene is added to wash the column. After washing,methanol is added to wash out the flavonoids before collecting 25 ml ofelute. The absorbance of elute is measured by using a UVspectrophotometer (at wavelength: 360 nm). Meanwhile, a Rutin(flavonoid-containing) standard solution is used to prepare a standardcurve for calculating the flavonoid amount in the sample.

Standard Curve Preparation:

5 mg Rutin (purchased from The National Institute for the Control ofPharmaceutical and Biological Products, Beijing, China) is added to 100ml methanol to make a 50 ug/ml standard solution. Different dilutions ofthe standard solution (0 ug/ml, 5 ug/ml, 10 ug/ml. 15 ug/ml, 20 ug/ml,and 25 ug/ml) are prepared. The absorbance of the solutions isdetermined using a UV detector at wavelength 360 nm. The standard curveis plotted from the results, using the following formula:

${X = \frac{A \times V_{2} \times 100}{V_{1} \times M \times 100}},$

where X=amount of flavonoids in the sample (mg/100 g); A=amount offlavonoids determined from the standard curve; M=mass of the sample (g);V₁=volume of the sample solution used in the test; V₂=total volume ofthe sample solution. X is corrected to 2 significant figures. For thisextraction process, flavonoids should not be less than 800 mg/100 g.

Example 3 Manufacturing of Rhodiola and Ginkgo Extract Supplements

Raw materials (both the herbal ingredients and the excipients) areapproved by the Quality Assurance Department before production iscarried out. The ingredient powders (prepared as described above) arefirst accurately weighed according to a master formula with anelectronic balance. The identity and quantity of each item to be weighedis verified. The weighed herbal ingredients and excipients are thenloaded into a fluid bed dryer (Model number: GCTB-30; Changzhou DelsonPharmaceutical Machinery, Changzhou, China). Air flow is adjusted sothat the powders are mixed sufficiently for 15-20 minutes. Amoisturizing agent (75% ethanol) is then sprayed into the fluid beddryer to allow the powder to granulate. During this process, theequipment inlet air temperature is maintained at 35-50° C. and theoutlet air temperature at 25-30° C. After forming 20 mesh size granules,the granules are dried at 50±2° C.

The dried granules are then screened and sifted with an 18 mesh sizestainless steel sieve by vibrating with screening machine (Model number:YK-160A; JiangYin Dry Equipment Co. Ltd., Jiangsu, China). Magnesiumstearate is used as a lubricant and is added at 0.2% of the total weightof the granules. The powder is then mixed in a 3D motion mixer for 15minutes. After being mixed sufficiently, 20 grams of the resultingpowder is sampled for QC using an in-process control test for moisture(should be less than 5%).

After passing the QC in-process control test, the granules aretransferred to a rotary tableting machine for tableting. The pressure,rotating speed and the tablet weight are adjusted so that each tabletweight reaches 0.4 g. The tablet fill weight and the intermediateproduct weight variation control range should be 0.4 g+7%. During thetableting process, 10 tablets are sampled and weighed for the total andindividual weights every 20 minutes to check for weight variation. Aftertableting, 60 tablets are sampled for weight variation. Twenty tabletsare subjected to a disintegration test (disintegrate within 30 minutes,or as required by target-country's regulations).

Film-coating of the tablets is carried out using a film-coating machine(Model number: GBB-600; Shanghai Zhong Lian Pharmaceutical Equipment,Shanghai, China). Film-coating solution is prepared by combining apre-mixed film-coating agent (prepared in accordance with the standardQ/WS-2693-2001 in the Guangdong Provincial Drug Approval Notice (2001) #934001) and 50% ethanol. The ratio between the pre-mixed film-coatingagent and 50% ethanol is 1:7. The amount of coating (by weight) shouldnot exceed 3-6% of the uncoated tablet weight. A sample of coatedtablets are subjected to a disintegration test (disintegrate within 60minutes, or as required by target-country's regulations). A sample oftablets may also be tested for ash content, heavy metal (arsenic, lead,mercury), microbes (total aerobic, E. coli, yeast and mold, pathogens),and/or pesticides.

The tablets are counted, filled into bottles and fitted with properclosures. The pack size is 60 tablets per bottle. During the internalpackaging process, 3 bottles will be sampled on 3 occasions to test forseal integrity, count size, closure fitness.

Filled bottles are affixed with labels imprinted with batch number andexpiration date. Labelled bottles are then sealed with transparent fullbody sleeve shrink wrap. 100 bottles are packed into 1 carton. 7 bottlesare sampled on three occasions to test for batch number and expirationdate, label position, shrink wrap position, fitness, smoothness,cleanliness and integrity. After passing the QC test, the product willbe released as finished products and properly stored. A schematicversion of this process of supplement manufacture is shown in FIG. 3.

Example 4 Rhodiola crenulata and Ginkgo biloba Enhances EndurancePerformance in Healthy Volunteers

Empirical evidence has implicated that many herbal medicines possessathletic performance-improving potentials. This study was designed todetermine whether the ingestion of an herbal supplement calledRhodiola-Gingko Capsule (RGC) would enhance endurance performance ofhealthy volunteers and change relevant hormones in a favorable manner.

Subjects and Estimation of Sample Size:

In order to minimize variations in demographic and baseline variables,the recruitment of subjects was limited to non-professional,college-level sportsmen who met the following criteria: 1) aged 18-22years; 2) had moderate-intensity exercises in a regular manner, definedas doing exercises 5-7 hours per week; and 3) whose maximal oxygenuptake (VO_(2max)) and body mass index (BMI) were 47-75 mL.min⁻¹.kg⁻¹and 18.5-26, respectively. Those who had unstable medical conditions, ahistory of alcohol or substance abuse within 1 year prior to the study,allergies to herbal medicines, needle phobia, or were currently underherbal or conventional medications were excluded from this study.

The present study was intended to detect a minimum 25% differencebetween RGC and placebo-treated groups in endpoint-to baseline change inVO_(2max), with a power of (1-β)=90% and a two-side level of α=0.05,using t-test or analysis of variance (ANOVA) model. Based on theseassumptions, about 30 subjects per group were needed and the numberincreased to nearly 35 per group when 15% of participants were expectedto be dropouts. Thus, seventy college-level sportsmen (18-22 years old)who had regular, moderate-intensity exercises were selected andrandomized to ingest RGC (4 capsules/day, 400 mg herbalextracts/capsule, n=35) or equivalent placebo (n=35) for 7 weeks underdouble-blind condition. Endurance exercise tests were conducted atbaseline, week 4, and endpoint.

Preparation of RGC and Placebo:

RGC and equivalent placebo used in the study were manufactured byIntegrated Chinese Medicine Holdings of Hong Kong, by which RGC has beenregistered as a dietary supplement in the State Food and DrugAdministration of China (registered number: G20060716; trademark:Sportchi™). RGC is formulated with Rhodiola crenulata and Ginkgo bilobaleaves in a ratio of 9:1 in dry weight. The identification andextraction of both herbs were processed as described in Pharmacopoeia ofthe People's Republic of China, PHARMACOPOEIA COMMISSION OF CHINA (ed.);Chemical Industry Press, 2005. The extracted powder of both herbs wasfully mixed and formed into dark orange, nontransparent capsules eachcontaining 270 mg mixed extractives. The placebo was prepared withstarchy powder in capsules identical to the active capsules in shape,size, and color.

To evaluate the quality of the preparation, the content of salidrosideand flavonoids contained in the active capsules was measured usinghigh-performance liquid chromatography (HPLC) and showed 23.80 mg/g and12.55 mg/g extracted powder, respectively, much higher than thestandards set in the Pharmacopoeia of the People's Republic of China,PHARMACOPOEIA COMMISSION OF CHINA (ed.); Chemical Industry Press, 2005.The content of heavy metals, insecticides, stimulants, narcotics, βblockers, diuretics, and steroid hormones was undetectable or below thestandards required.

Study Design and Procedures:

A double-blind, randomized, placebo-controlled design was used in thestudy. Participants were randomly assigned to receive RGC or placebosupplement in the ratio of 1:1 for 7 weeks in double-blind fashion,i.e., either participants or investigators (including capsuledispensers) were unaware of supplement conditions during the entirecourse of the study (supplement codes were kept with the manufacturerand not disclosed until the completion of data input). Each participantwas required to take 4 capsules per day (2 after breakfast and 2 aftersupper). Assigned investigators directly dispensed capsules to eachparticipant and monitored their ingestion on a daily basis. Participantswere also asked to record their daily dietary, exercise and sleeppatterns as well as incidence of adverse events. Endurance exercisetests were conducted at baseline (pretreatment), week 4, and endpoint(after completion of the treatment). All data was recorded in the datareport form designed.

Endurance Exercise Tests:

Each subject was asked to perform a set of endurance exercise testsconsisting of a 30-sec fixed workload test and a maximal incrementaltest at baseline (before treatment), week 4, and endpoint (aftercompletion of treatment). The subjects were instructed to avoidstrenuous exercises for 24 h before each test session and to arrive atthe exercise testing laboratory in the rested and fully hydrated state.Food, caffeine, and alcohol intake was prohibited for 3 h beforetesting. The laboratory was maintained at 19-21° C. and 40-60% relativehumidity during exercise testing. Each subject was tested at the sametime on testing days to control circadian and diurnal influences.

30-sec fixed workload test: The test was conducted on a cycloergometer(Monark 834) to measure parameters associated with the power output. Thetest started with a warm-up without workload, through which 150-160beat/min of heart rate was required to be achieved (3-5 min). Followingthis warm-up, subjects performed maximal cycling exercise with a fixedworkload equal to 7.5% of body weight for 30 sec. Maximal power output(W_(max)), minimum power output (W_(min)), mean power output (W_(mean))and drop rate of the power output (W_(drop)) were measured based on a5-sec interval data record.

Maximal incremental test: About 30 minutes after the completion of thefixed workload test, subjects started the maximal incremental test. Timeto exhaustion and VO_(2max) were measured in the test. The test wasconducted on a motorized treadmill running machine (h/p/cosmos, Germany)using a standard Bruce Protocol (Baba et al., J. Am. Coll. Cardiol.286:1567-1572, (1996)). Briefly, the slope of the treadmill was set at10% with a speed of 1.7 mph in the initial 3 minutes of the test. Thetwo parameters were then increased by 2% and by 0.5-0.9 mph every 3minutes, respectively. Gas exchange of subjects' breath was analyzedcontinuously using an automated gas exchange analyzer (Max II, USA)through a facemask. Heartbeat was measured using a wireless heart ratemonitoring method (Polar, Sweden). The test was stopped and exhaustivetime and VO_(2max) were recorded when any two of the following maximalcriteria occurred: VO₂ plateau, respiratory exchange ratio (RER)>1.15,or heart rate >180 beat/min; or when the subject was exhausted and couldnot maintain the imposed treadmill speed.

Determination of Blood Testosterone and Cortisol Under RestingCondition:

Two 8-ml blood samples were collected under resting condition in themorning (7:00-8:00 AM), before meal at baseline and endpoint,respectively. Sera were immediately separated and stored at −80° C. forassay. Serum concentrations of testosterone (T) and cortisol (C) weremeasured using radioimmunoassay (RIA) and ratios of testosterone(nmol/L) to cortisol (μmol/L) was calculated. In order to excludeinter-assay variations, all samples were processed in one “batch” withthe same assay kit under the same condition.

Statistical Analysis:

In order to exclude the influences of basal variations in enduranceexercises, changes in endurance variables at week 4 and endpoint frombaseline were used for statistical analysis. Baseline variables wereanalyzed using Student t-test. Two-way repeated measure analysis ofvariance (ANOVA) was used to detect main effects of supplement andtreatment time on endurance and hormone parameters. Pairwise multiplecomparisons were further conducted with Students-Newman-Keuls method ifsignificant main effects were present. Data were expressed asmean±standard deviation (SD) unless otherwise indicated. All tests weretwo-sided and statistical significance was defined as p<0.05.

Subject Disposition and Characteristics:

A total of 70 sportsmen participated in the study and were randomlyassigned to placebo (n=35) or RCG supplement (n=35). One participantinitially assigned to placebo was removed from data analysis because hisage did not reach the criterion (only 17 years old). Two participantsallocated to RCG were excluded due to BMI value (26.4) higher than thecriterion and incomplete endurance exercise tests, respectively. Nearly95% of participants had moderate-intensity exercise for 1-2 hours inmost days during the study. Nearly 82-85% of participants of each groupslept for 6-8 hours nightly during the study. Almost all participantsdefined as their dietary pattern for mixed vegetable and meat. Baselinedemographic and endurance exercise variables are shown in Table 1 andTable 2. No statistically significant differences were observed onbaseline variables between the two groups.

TABLE 1 Demographic characteristics of subjects Variable Placebo (n =34) RGC (n = 33) p value ^(a) Age (yr) 20.0 (1.3) 19.9 (1.0) 0.748Height (cm) 175.7 (4.6)  174.7 (4.5)  0.367 Weight (kg) 67.2 (7.1) 65.2(6.1) 0.223 BMI 21.7 (1.7) 21.3 (1.9) 0.379 ^(a) Student t-test was usedto detect statistical significance between the two groups.

TABLE 2 The effects of 7-week supplement with placebo and RGC on changesin endurance exercise tests at week 4 and endpoint from baseline valuesin college-level sportsmen Baseline Changes at week 4 Changes atendpoint Variables Placebo RGC p value^(a) Placebo RGC Placebo RGC^(b)Time to exhaustion (sec) 801.6 (71.7) 794.1 (65.1) 0.654 −7.5 (39.0) 7.1(36.4) −9.7 (38.2) 10.3 (46.2)* VO_(2max) (mL · min⁻¹ · kg⁻¹) 62.0 (6.1)59.5 (6.5) 0.101 1.4 (6.7) 3.2 (6.1) 0.4 (6.4) 3.9 (5.2)* peak poweroutput 10.1 (1.7) 10.0 (0.8) 0.963 −0.1 (1.6) −0.2 (1.2) 0.0 (1.6) 0.0(1.5) (W · kg⁻¹) Minimum power output 5.8 (0.9) 5.5 (0.9) 0.237 −0.4(1.0) 0.0 (1.3) −0.3 (1.0) −0.3 (1.0) (W · kg⁻¹) Average power output7.7 (0.7) 7.6 (0.5) 0.449 −0.2 (0.9) −0.1 (1.0) −0.2 (0.7) −0.2 (1.0) (W· kg⁻¹) power drop (W · sec⁻¹ · 0.2 (0.1) 0.1 (0.1) 0.183 0.0 (0.2) 0.0(0.1) 0.0 (0.2) 0.0 (0.1) kg⁻¹) ^(a)Student t-test was used to detectstatistical differences in basal values between placebo- (n = 34) andRGC-ingested subjects (n = 33). ^(b)Two-way repeated measure ANOVA wasconducted and followed by multiple group comparisons. *P < 0.05 vs.placebo group at the same time point.

Effects on Endurance Exercise Parameters:

The results of endurance exercise tests are summarized in Table 2.Two-way repeated measure ANOVA revealed a significant effect oftreatment (F_(1,66)=4.480, P=0.038), but not treatment time(F_(2,65)=0.01, ⁻P=0.995), on changes in time to exhaustion frombaseline. Multiple comparisons further revealed that the extension oftime to exhaustion in RGC-treated subjects was significantly greaterthan placebo at endpoint (P=0.012).

There were significant effects of both treatment (F_(1,66)=4.081,P=0.047) and time (F_(2,65)=6.955, P=0.001) on increases in VO_(2max)from baseline. Multiple group comparisons further revealed that theamplitude of the increased VO_(2max) in RGC-ingested subjects wassignificantly greater than placebo at endpoint (P=0.003). The increasedamplitudes at week 4 and at endpoint were also significantly greaterthan baseline in RGC-ingested subjects (P<0.001), but not inplacebo-ingested subjects (P>0.449) (FIG. 4).

Either treatment or time had no significant effects on changes inW_(max) (treatment: F_(1,66)=0.101, ⁻¹=0.751; time: F_(2,65)=0.562,P=0.576), W_(min) (treatment: F_(1,66)=1.225, P=0.272; time:F_(2,65)=2.056, P=0.108), W_(mean) (treatment: F_(1,66)=0.063, P=0.803;time: F_(2,65)=2.435, P=0.092), or W_(drop) (treatment: F_(1,66)=1.508,P=0.224; time: F_(2,65)=0.745, P=0.477).

Effects on resting hormone levels: The effects on serum testosterone,cortisol concentrations and ratio of testosterone (nmol/L) to cortisol(μmol/L) are illustrated in Table 3 and FIG. 5.

TABLE 3 The effects of 7-week supplements with placebo and RGC on serumhormones in college-level sportsmen^(a) Baseline Endpoint VariablesPlacebo RGC Placebo RGC Testosterone 38.1 (8.4)  37.4 (9.2)  41.2(10.6)  38.4 (11.0) (T, nmol/L) Cortisol 0.6 (0.2) 0.7 (0.2) 0.8(0.2)^(#) 0.7 (0.3) (C, μmol/L) T/C ratio 64.6 (23.3) 56.2 (14.8) 54.5(17.1)^(#) 61.3 (36.7) ^(a)Two-way repeated measure ANOVA was conductedto detect statistical significance in placebo-(n = 34) and RGC-ingestedsubjects (n = 33), followed by multiple group comparison. ^(#)p < 0.05vs. the same group at baseline.

The two groups of subjects had similar baseline serum concentrations oftestosterone (t value=0.329, P=0.743), cortisol (t value=1.189,P=0.239), and T/C ratio (t value=1.758, P=0.083). No significant effectswere observed on testosterone by either treatment (F_(1,66)=0.765,P=0.385) or time (F_(2,65)=2.565, P=0.114). Time factor had asignificant effect on cortisol (F_(1,65)=10.845, P=0.002), but treatmentfactor did not (F_(1,65)=0.003, P=0.957). Multiple comparisons furthershowed that the mean cortisol level was significantly higher at endpointthan baseline in placebo-ingested subjects (P<0.001), but similarbetween the two time points in RGC-ingested subjects (P=0.294). Asignificant effect on the ratio was observed on time (F_(1,65)=5.096,P=0.027), but not on treatment (F_(1,65)<0.001, P=0.983). Multiplecomparisons displayed a significant baseline-to-endpoint decrease in theratio in placebo-ingested subjects (P=0.029), but not in RGC-ingestedsubjects (P=0.339).

Adverse Events:

There were 9 subjects (4 in placebo and 5 in RGC) who experiencedtransient sleepiness in the initial phase of treatment. No other adverseevents were reported.

Summary:

The present study represents a methodologically rigorous investigationevaluating the efficaciousness of the herbal supplement prepared from acombination of Rhodiola crenulata and Ginkgo biloba in improvingendurance exercise performance in comparison with placebo. In order toheighten the sensitivity and validity of the study, a highly uniformpopulation was defined, characterized by the same gender, similardemographic, exercise, dietary and sleep patterns.

The present study found that the ingestion of RGC for 7 weeks resultedin an approximately 10-fold greater increase of VO_(2max) from thebaseline compared to placebo. Moreover, while placebo-treated subjectsshowed a shorter time to exhaustion at endpoint (nearly 10-secondshorter than baseline), RGC-treated subjects displayed a remarkableextension of exhaustive time (about 10 seconds increased from baseline),yielding a significant difference between the two groups at endpoint(see Table 3 and FIG. 4), although indices associated with power outputwere unaffected. On the other hand, it is well known that cortisolexcess and a decreased ratio of testosterone to cortisol are importantindicators for overtraining and fatigue in endurance exercises (Urhausenand Kindermann, Sports Med. 32:95-102, (2002); Reilly and Ekblom, J.Sports Sci. 23:619-627, (2005)). In the present study, we found that,while placebo-ingested subjects displayed the significantly increasedlevel of blood cortisol and the significantly decreased ratio oftestosterone to cortisol under resting condition, the two hormoneindices remained unchanged in RGC-ingested subjects following 7 weeks oftreatment. These findings suggest that RGC possesses endurance-enhancingand anti-fatigue effects, protecting against declines in enduranceperformance; and the effects are largely achieved by increasing oxygenconsumption.

Example 5 Cellular Effects of Rhodiola crenulata and Ginkgo biloba

To determine whether the combination of Rhodiola crenulata and Ginkgobiloba extracts has effects at the cellular level, in vitro tests wereperformed. RG, composed of 90% Rhodiola crenulata with 10% Ginkgobiloba, (batch #: 071001, Hong Kong Health Care Association Ltd),Rhodioloa Crenulata (batch #: HJ070202-H, Hong Kong Health CareAssociation Ltd), Ginkgo biloba (bath #: 060802, Hong Kong Health CareAssociation Ltd), and Vitamin C (Sigma, Co.) were used to treat L6 ratskeletal cells (L6) in DMEM media.

The data discussed below is represented by mean±SEM. Statisticalanalysis was performed using the two-tailed Student's t test fordifference between samples. A difference with p<0.05 was consideredstatistically significant.

A) MTT Assay

L6 cells were cultured in 96-well tissue culture plates (1×10⁴cell/well) with 10% FBS for 24 hours. Serum-free medium was then usedfor another 24 hours prior to treatment with RGC, Rhodiola crenulataextract, or Ginkgo biloba extract. Cell viability and proliferation weremeasured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and 5-bromo-2′-deoxyuridine (BrdU) (Roche Diagnostics,USA) incorporation assays, respectively.

L6 cells were maintained in DMEM medium supplemented with 10% fetal calfserum, 100 U/ml penicillin and 100 U/ml streptomycin in a humidatmosphere of 5% CO2 and 95% air at 37° C. The L6 cells were plated in96-well plates (5000 cell/well) for 24 hours. Then the serum-free mediumwas used and cells incubated for another 12 hours. Sequentially, cellswere pretreated with various concentrations (20 μg/ml, 40 μg/ml, 80μg/ml) of RG, Rhodiola Crenulata, or Ginkgo biloba, respectively, orVitamin C (80 μg/ml) for 12 hours, followed by exposure to an ischemicsolution (EBSS) and placed in an anaerobic chamber (Diagnostic System)for 1 hour. The control cells were incubated without treatment for 1hour. After 1 hour exposure to hypoxic and ischemic environments, 100 μlMTT (0.5 mg/ml in PBS) was added to each well and the cells wereincubated at 37° C. for 4 h. The MTT solution was prepared by dissolving250 mg of MTT in 50 ml PBS (0.01 mol/L, PH7.4) to prepare 0.5% MTTstaining solution. The solution was then filtered through Milliporemembrane and stored at 4° C.

The supernatants were removed carefully and 100 μl of dimethyl sulfoxide(DMSO) was added to each well to dissolve the precipitate. Theabsorbance at 570 nm was measured with a Infinite M200 microplate reader(TECAN) to evaluate the vitality of cells and as shown in Table 4 andFIG. 6. The cell proliferation in RGC, Rhodiola crenulata and Ginkgobiloba groups were significantly higher than that in the vehicle groupunder the hypoxic condition. The effect was dose-dependent. Thisindicated that RG, Rhodiola crenulata and Ginkgo biloba leaf extract hasa protective effect to the rat skeletal muscle cells in hypoxiccondition. RG (80 ug/ml) and Rhodiola crenulata (40 ug/ml) had asignificant protective effect to the hypoxic rat skeletal muscle cellwhen compared with the Ginkgo biloba (40 ug/ml) (p<0.01).

TABLE 4 Cell proliferation in hypoxic conditions^(a) 0 μg/ml 20 μg/ml 40μg/ml 80 μg/ml Vehicle 27% ± 1.3% N/A N/A N/A RG N/A  44% ± 1.08%** 52%± 5.17%**  60% ± 5.87%** Rhodiola N/A 43% ± 8.14%* 62% ± 4.19%** 51% ±1.9%** crenulata Ginkgo biloba N/A 33% ± 4.9%*  38% ± 4.8%*  36% ±6.0%*  Positive Control N/A N/A N/A 41% ± 3.2%** ^(a)The cell survivalrate under hypoxic condition. Cell viability is measured by the MTTassay. Data is presented as means ± SD. *significantly different fromvehicle at P < 0.05; **significantly different from vehicle at P < 0.01.

B) LDH Assay

Trypan blue is one of several stains used for dye exclusion procedurefor viable cell counting. The primary assessment of cell viability inthis study is based on measurement of lactate dehydrogenase enzyme (LDH)leakage from the cells into the medium; an indicator of relative cellviability, using TOX-7 LDH based in vitro toxicology assay kit (Sigma,St Louis, USA). The LDH assay is measured spectrophotometrically at 490nm.

L6 cells were maintained in DMEM medium supplemented with 10% fetal calfserum, 100 U/ml penicillin and 100 U/ml streptomycin in a humidatmosphere of 5% CO2 and 95% air at 37° C. L6 cells were plated in12-well plates (4×10⁴ cell/well) for 24 hours. Then the serum-freemedium was used and cells incubated for another 12 hours. Sequentially,cells were pretreated with various concentrations (20 μg/ml, 40 μg/ml,and 80 μg/ml) of RG, Rhodiola Crenulata, or Ginkgo biloba, respectively,or Vitamin C (80 μg/ml) for 12 hours, followed by exposure to ischemicsolution (EBSS) placed in an anaerobic chamber (Diagnostic System) for 1hour. The control cells were incubated without treatment for 1 hour aswell. Subsequently, the supernatants were removed and treated cells werewashed with PBS followed 100 μl or lysis buffer. The cells werecollected and centrifuged at 5000 r/min for 10 min at 4° C., thesupernatant gathered and protein concentration determined using a BCAprotein quantitative assay (BCA Protein Quantitative Analysis kit,available from Shanghai Biocolor Bioscience & Technology Company).

To prepare the protein standard curve for the BCA protein quantitativeassay, 0, 0.5 μl, 1 μl, 2 μl, 4 μl, 6 μl, 8 μl, and 10 μl of dilutedstandard protein (0.5 mg/ml) was pipetted into wells and distilled wateradded to have a final volume of 20 μl for each well. Working solutionwas freshly prepared of which 200 ul was added in each well followed byincubated at 37° C. for 30 min. The absorbance at 562 nm was measuredwith a Infinite M200 microplate reader (TECAN) to calculate the standardcurve. To determine the optimal sample volume for the BCA proteinquantitative assay, various volume samples were diluted to the samefinal volume 20 μl then tested using BCA protein quantitative assay andthe optimal sample volume chosen, 4 μl, was used in the BCA proteinassay for the samples.

The optimal sample volume for LDH assay was determined by testingvarious volume samples that were diluted to the same final volume of 20μl (see Tables 5 and 6). A LDH quantitative assay (commerciallyavailable LDH Assay Kits can be used) was then performed for each. Thesample testing tubes were mixed well then placed in a water bath at 37°C. for 15 min. Then 50 μl dinitro-phenyl-hydrazine was added to eachtube, mixed, and then placed in a water bath at 37° C. for 15 min. Next,500 ul 0.4 mol/LNaOH was added to each tube and then left at roomtemperature for 3 min before detecting the absorbance at 440 nm.Volume-absorbance curve was acquired with sample volume as the X-axiswhile the absorbance as the Y-axis, and the volume between the linearrange was chosen. 2 μl was selected as the best testing volume and usedin for the LDH Assay.

TABLE 5 LDH pilot testing tubes Sample (μl) 0 1 2 4 8 12 Basis buffer(50 μl) 50 50 50 50 50 50 Distilled water (μl) 20 19 18 16 12 8 CoenzymeI (μl) 10 10 10 10 10 10

TABLE 6 LDH pilot testing vehicle tubes Sample (μl) 0 1 2 4 8 12 Basisbuffer (50 μl) 50 50 50 50 50 50 Distilled water (μl) 20 19 18 16 12 8Coenzyme I (μl) 10 10 10 10 10 10

After optimal BCA assay volume and optimal LDH assay volumes weredetermined, the protein concentration and LDH activity for the samplesof the various treatment groups (20 μg/ml, 40 μg/ml, or 80 μg/ml of RG,Rhodiola Crenulata, or Ginkgo biloba, respectively, and 80 μg/ml ofVitamin C) where the sample volumes were as shown in Table 7. The LDHactivity assay was then performed where each sample was mixed and placedin a water bath at 37° C. for 15 min before 50 μldinitro-phenyl-hydrazine was added to each tube. The samples were thenmixed and placed in a water bath at 37° C. for 15 min. Next, 500 ul 0.4mol/LNaOH was added to each tube and then left at room temperature for 3min before detecting the absorbance at 440 nm. The LDH activity wascalculated according to the formula: LDH activity (U/g protein)=(TestingOD−Testing vehicle OD)/(Standard OD−Standard vehicle OD)×Standardconcentration/protein concentration (g of protein/ml) and is shown inTable 8 and FIG. 7.

TABLE 7 LDH activity assessment St St vehicle Testing vehicle TestingBasis buffer (μl) 50 50 50 50 2 μmol/Lpyruvate (μl) 2 Distilled water(μl) 10 12 10 Sample (μl) 2 2 Coenzyme I (μl) 10

As shown in Table 8 and FIG. 7, the LDH leakage rate in RG, Rhodiolacrenulata and Ginkgo biloba groups were significantly lower than that inthe vehicle group under the hypoxic condition. This indicated that RG,Rhodiola crenulata and Ginkgo biloba leaf extract has a protectiveeffect to the rat skeletal muscle cells in the hypoxic condition. RG (40ug/ml) has significant protective effect to the hypoxic rat skeletalmuscle cell when compared with the Rhodiola crenulata (40 ug/ml)(p<0.05) and Ginkgo biloba (40 ug/ml) (p<0.01), respectively. Rhodiolacrenulata (40 ug/ml) also has significant protective effect to thehypoxic rat skeletal muscle cell when compared with the Ginkgo biloba(40 ug/ml) (p<0.01).

TABLE 8 LDH Leakage Rates^(a) 0 μg/ml 20 μg/ml 40 μg/ml 80 μg/ml RG N/A26% ± 2.3%* 6% ± 1.7%** 18% ± 1.1%** Rhodiola N/A  16% ± 1.7%** 9% ±2.9%**  9% ± 2.22%** crenulata Ginkgo biloba N/A 20% ± 3.1%* 18% ±2.1%**  23% ± 1.8%*  Vehicle 24% ± 2.6% N/A N/A N/A ^(a)The LDH leakageof L6 cells under hypoxic condition. The LDH leakage is measured by LDHassay. Data is presented as means ± SD. *significantly different fromvehicle at P < 0.05; **significantly different from vehicle at P < 0.01.

C) SOD Antioxidant Assay

Cells were homogenized in ice-cold phosphate buffer (50 mmol/L, pH 7.4)to make 10% w/v homogenate with a motor-driven Potter-Elvejhem glasshomogenizer at 0-4° C. The homogenate will be then centrifuged at 300 gfor 10 min at 4° C. to remove intact cells and debris. The remainingsupernatant was divided into 2 parts for different enzyme assays. ForSOD assays, the supernatant will be centrifuged at 2300 g for a further10 min at 4° C. After centrifugation, the pellets will be discarded andthe supernatants were used for the assessment of enzyme activity. Theprocedure was carried out on ice or at 0-4° C. Reagents for the assaywere obtained through a commercially available SOD Assay Kit. Theoptimal sample volumes for the BCA quantitative assay and the SOD assaywere determined as described in the LDH assay.

After determination of optimal sample volumes, a pilot study wasperformed using the volumes as shown in Table 9. The samples were mixedand placed in a water bath at 37° C. for 40 min. A chromogenic agent(200 μl) was then added, the samples mixed, then left at roomtemperature for 10 min before absorbance at 550 nm was detected. TheInhibition ration was then calculated using the formula: inhibitionratio=(ODn−OD1)/OD1×100%. The inhibition ratio between 48%-50% wasconsidered as the optimal sample volume, and thus, 10 μl was selectedfor use for the SOD assays. The protein concentration was measured usingthe BCA protein quantitative assay.

TABLE 9 SOD pilot study Sample (μl) 1 2 4 6 8 10 12 Water (μl) 1 2 4 6 810 12 Reagent 1 (μl) 100 100 100 100 100 100 100 Reagent 2 (μl) 100 100100 100 100 100 100 Reagent 3 (μl) 100 100 100 100 100 100 100 Reagent 4(μl) 100 100 100 100 100 100 100

After 10 μl was selected for use for the SOD assays, SOD activity assayswere performed for the samples of the various treatment groups (20μg/ml, 40/g/ml, or 80 μg/ml of RG, Rhodiola Crenulata, or Ginkgo biloba,respectively, and 80 g/ml of Vitamin C) where the sample volumes were asshown in Table 10. The samples were mixed and placed in a water bath at37° C. for 40 min. A chromogenic agent (200 μl) was then added, thesamples mixed, then left at room temperature for 10 min beforeabsorbance at 550 nm was detected. The SOD enzyme activity was thencalculated according to the formula: T-SOD activity(U/gprot)=(ControlO.D.−Testing O.D.)/Control O.D./50%×total volume/sample volume/proteinconc.(gprot/ml).

TABLE 10 SOD activity assay T-SOD control T-SOD testing Reagent 1 (μl)100 100 Distilled water (μl) 10 10 Sample (μl) 10 10 Reagent 2 (μl) 1010 Reagent 3 (μl) 10 10 Reagent 4 (μl) 10 10

The results as shown in Table 11 and FIG. 8 reveal that SOD enzymaticactivity in RG, Rhodiola crenulata and Ginkgo biloba groups aresignificantly higher than that in the Vehicle group.

TABLE 11 SOD Enzymatic Activity 0 μg/ml 20 μg/ml 40 μg/ml 80 μg/ml RGN/A 18.83% ± 0.47%*  18.50% ± 0.27%** 20.26% ± 0.54%** Rhodiola N/A16.48% ± 1.20%* 19.61% ± 1.19%* 19.98% ± 0.87%*  crenulata Ginkgo bilobaN/A 17.09% ± 1.11%* 19.79% ± 1.05%* 20.36% ± 0.26%** Vc N/A N/A N/A20.00% ± 0.08%*  Vehicle 16.58% ± 0.31%  N/A N/A N/A Vehicle 20.98% ±0.11%** N/A N/A N/A ^(a)The SOD activity of L6 cells under hypoxiccondition. The SOD activity is measured by SOD assay. Data is presentedas means ± SD. *significantly different from vehicle at P < 0.05;**significantly different from vehicle at P < 0.01.

Conclusions:

Chinese herbal extract RG (Rhodiola crenulata and Ginkgo biloba)enhances cell growth and proliferation in normoxic and hypoxicconditions. The study adopts MTT, LDH and SOD calorimetric assays. TheMTT assay shows that the cell viability of cells treated with RG arehigher than that of vehicles and negative controls under hypoxicconditions and the result shows dose-dependence. The LDH assay showsthat the LDH leakage of cells treated with RG is lower than the Vehiclegroup. The SOD assay shows that the SOD activity of cells treated withRG are higher than the Vehicle group. The study shows that RG canpromote the proliferation of the rat skeletal muscle cells and haveanti-hypoxia and protective effects. Furthermore, the study also showsthat RG has a much better effect than Gingko biloba alone and a greatereffect than Rhodiola crenulata alone.

Example 6 Cellular Effects of Rhodiola crenulata and Ginkgo biloba onHuman Cells

To determine whether the combination of Rhodiola crenulata and Ginkgobiloba extracts have effects at the cellular level in human cells, invitro tests are performed. The tests are conducted by adding thecombined extracts, the extracts individually, or no extract to humanskeletal muscle cells in vitro and determining effects of severalcellular functions. The expression of vascular endothelial growth factor(VEGF), myoglobin and citrate synthase genes, and SOD enzymaticactivity, and cell proliferation and growth are measured under normoxicand hypoxic conditions. Increase in the gene expression of VEGF andmyoglobin indicate improved capability to provide oxygen to the cells.Alternation of the citrate synthase encoding gene, a commonly usedmarker for the presence of intact mitochondria in muscle cells,indicates muscle adaptation to hypoxic conditions. Alternation(increase) of SOD enzymatic activity from the SOD antioxidant assaysuggests increase in the oxidative capacity of the cells under hypoxicconditions.

Example 7 Cellular Effects of Rhodiola crenulata and Ginkgo bilobaCombinations

To determine whether different ratios of extracts of Rhodiola crenulataand Ginkgo biloba have differing effects on cells, the followingexperiments were performed. Rat skeletal muscle cells L6 cells weremaintained in DMEM medium supplemented with 10% fetal calf serum, 100U/ml penicillin and 100 U/ml streptomycin in a humid atmosphere of 5%CO2 and 95% air at 37° C. Rat Skeletal Muscle Cells L6 cells were platedin 6-well plates (1×10⁵ cell/well) for 24 hours. Serum-free medium wasthen used and cells incubated for another 12 hours. Sequentially, cellswere pretreated with various concentrations groups for 12 hours. Thegroups were as follows: Group 1—80% Rhodiola crenulata+20% Ginkgo biloba(e.g. total 80 ug/ml), Group 2—80% Rhodiola crenulata (e.g. total 64ug/ml), Group 3—100% Rhodiola crenulata (e.g. total 80 ug/ml), Group 4(“RG” group)—90% Rhodiola crenulata+10% Ginkgo biloba (e.g. total 80ug/ml); Group 5—90% Rhodiola crenulata (e.g. total 72 ug/ml), Group6—Vit. C as control group, Group 7—Vehicle, Group 8—Control.

Each group was exposed to an ischemic solution (EBSS) and placed in ananaerobic chamber (Diagnostic System) for 2 hours. The control cellswere incubated without treatment for 2 hours as well. The supernatantswere then removed and the treated cells were washed with PBS followed100 μl of lysis buffer. The cells were collected and centrifuged at 5000r/min for 10 min at 4° C. The supernatant was then gathered and theprotein concentration determined was using the BCA protein quantitativeassay, as described in Example 5. The LDH activity (FIG. 9) and SODactivity (FIG. 10) were determined using methods as described in Example5.

As shown in FIG. 9, the LDH leakage rate in Groups 1 to 6 aresignificantly lower than that in the Vehicle group under the hypoxiccondition. In addition, Group 4 has a significant protective effect tothe hypoxic rat skeletal muscle cell when compared with the other fivegroups. As shown in FIG. 10, SOD enzymatic activity in Group 3 and group4 are significantly higher than that in Vehicle group. In addition, SODenzymatic activity in group 4 is significantly higher than those fourgroups except group 5. This in-vitro study showed that group 4 (RG) havea significantly better anti-hypoxic effect than the other five groups inthe rat L6 skeletal muscle cells.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

1. A composition comprising an herbal extract of Rhodiola crenulata andan herbal extract of Ginkgo biloba, wherein the herbal extract ofRhodiola crenulata comprises at least about 0.40%, at least about 0.50%,at least about 0.60%, at least about 0.70%, at least about 0.80%, or atleast about 0.90% w/w salidroside based on the total weight of theherbal extract.
 2. The composition of claim 1, further comprising one ormore excipients.
 3. The composition of claim 2, wherein the compositionis a solid.
 4. The composition of claim 2, wherein the composition is aliquid.
 5. The composition of claim 1, wherein the herbal extract ofGinkgo biloba comprises at least about 5.0%, at least about 6.0%, atleast about 7.0%, at least about 8.0%, at least about 9.0%, at leastabout 10.0%, at least about 11.0%, at least about 12.0%, at least about13.0%, or at least about 14.0% w/w flavonoids based on the total weightof the herbal extract.
 6. The composition of claim 1, wherein the herbalextract of Rhodiola crenulata comprises about 5-95% w/w of the totalweight of the composition.
 7. The composition of claim 1, wherein theherbal extract of Ginkgo biloba comprises about 5-50% w/w of the totalweight of the composition.
 8. The composition of claim 1, wherein theherbal extract of Rhodiola crenulata comprises about 50-95% w/w and theherbal extract of Ginkgo biloba comprises about 5-50% w/w of the totalweight of the composition.
 9. The composition of claim 1, wherein theherbal extract of Rhodiola crenulata is about 90% w/w and the herbalextract of Ginkgo biloba is about 10% w/w of the total weight of thecomposition.
 10. The composition of claim 2, wherein the herbal extractof Rhodiola crenulata is about 45-70% w/w and the herbal extract ofGinkgo biloba is about 5-10% w/w of the total weight of the composition.11. A method for improving or restoring blood circulation in the body ofa mammal, comprising: administering to the mammal in need thereof aneffective amount of the composition of claim
 1. 12. The method of claim11, wherein said administration is oral.
 13. The method of claim 11,wherein the composition is administered to a human at a dose of about1-3200 mg/day, about 400-2000 mg/day, about 800-1600 mg/day, about1200-1600 mg/day, or about 1000 mg/day.
 14. The method of claim 11,wherein the composition is orally administered to a human at a dose ofabout 1-100 mg/kg, about 1-60 mg/kg, about 1-30 mg/kg, about 1-15 mg/kg,aboutl-6 mg/kg or about 1-5 mg/kg once or twice a day.
 15. A method forpromoting mental acuity in a mammal comprising: administering to themammal in need thereof an effective amount of the composition ofclaim
 1. 16. The method of claim 15, wherein said administration isoral.
 17. The method of claim 15, wherein the composition isadministered to a human at a dose of about 1-3200 mg/day, about 400-2000mg/day, about 800-1600 mg/day, about 1200-1600 mg/day, or about 1000mg/day.
 18. The method of claim 15, wherein the composition is orallyadministered to a human at a dose of about 1-100 mg/kg, about 5-50mg/kg, about 10-30 mg/kg, about 20-25 mg/kg, about 1-6 mg/kg or about1-5 mg/kg once or twice a day.
 19. A kit comprising at least one dose ofan effective amount of the composition of claim
 1. 20. The kit of claim19, further comprising instruction of how to use the kit to improve orrestore blood circulation, promote mental acuity, reduce fatigue, orpromote aerobic or anaerobic performance.
 21. The kit of claim 19,wherein more than one dose of an effective amount of the composition ofclaim 1 is included.
 22. The kit of claim 21 further comprising adispensing cup or a dispensing spoon.
 23. A method for reducing fatiguein a mammal comprising: administering to the mammal in need thereof aneffective amount of the composition of claim
 1. 24. A method forpromoting aerobic performance in a mammal comprising: administering tothe mammal in need thereof an effective amount of the composition ofclaim
 1. 25. The method of claim 24, wherein said administration isoral.
 26. The method of claim 24, wherein the composition isadministered to a human at a dose of about 1-3200 mg/day, about 400-2000mg/day, about 800-1600 mg/day, about 1200-1600 mg/day, or about 1000mg/day.
 27. The method of claim 25, wherein the composition is orallyadministered to a human at a dose of about 1-100 mg/kg, about 1-60mg/kg, about 1-30 mg/kg, about 1-15 mg/kg, about 1-6 mg/kg or about 1-5mg/kg once or twice a day.